Characterization of a hepatitis C virus-like particle vaccine produced in a human hepatocyte-derived cell line

Abstract

An effective immune response against hepatitis C virus (HCV) requires the early development of multi-specific class 1 CD8 + and class II CD4 + T-cells together with broad neutralizing antibody responses. We have produced mammalian-cell-derived HCV virus-like particles (VLPs) incorporating core, E1 and E2 of HCV genotype 1a to produce such immune responses. Here we describe the biochemical and morphological characterization of the HCV VLPs and study HCV core-specific T-cell responses to the particles. The E1 and E2 glycoproteins in HCV VLPs formed non-covalent heterodimers and together with core protein assembled into VLPs with a buoyant density of 1.22 to 1.28 g cm À3 . The HCV VLPs could be immunoprecipited with anti-ApoE and anti-ApoC. On electron microscopy, the VLPs had a heterogeneous morphology and ranged in size from 40 to 80 nm. The HCV VLPs demonstrated dose-dependent binding to murine-derived dendritic cells and the entry of HCV VLPs into Huh7 cells was blocked by anti-CD81 antibody. Vaccination of BALB/c mice with HCV VLPs purified from iodixanol gradients resulted in the production of neutralizing antibody responses while vaccination of humanized MHC class I transgenic mice resulted in the prodution of HCV core-specific CD8 + T-cell responses. Furthermore, IgG purified from the sera of patients chronically infected with HCV genotypes 1a and 3a blocked the binding and entry of the HCV VLPs into Huh7 cells. These results show that our mammalian-cell-derived HCV VLPs induce humoral and HCV-specific CD8 + T-cell responses and will have important implications for the development of a preventative vaccine for HCV.

A Hepatitis C Virus DNA Vaccine Encoding a Secreted, Oligomerized Form of Envelope Proteins Is Highly Immunogenic and Elicits Neutralizing Antibodies in Vaccinated Mice

“…All groups immunized with the DNA vaccines encoding E1E2 heptamers induced significant IFN-γ responses compared to animals immunized with the monomeric DNA constructs as determined by ELISpot. The number of SFUs detected in the E1/E2-specific ELISpot reported in this study compared favorably with those reported in other studies using viral vectors (76,77,90), DNA (91), or VLPs (67,91).…”

“…The HCV neutralization assay to measure the inhibition of HCV VLP binding and entry into Huh7 cells was performed as described previously (66,67…”

“…The purified sE1E2 poly-His-tagged fusion protein described above constituted the E1E2 protein boost. The production and purification of the genotype 1 (gt1) HCV VLPs have been described elsewhere (66,67). As shown above in Figures 3A,B, the antibody response plateau was reached following four DNA vaccinations, therefore mice were vaccinated with four doses of p-sE1-IMX313P + p-sE2-IMX313P or p-sE1E2-IMX313P, before receiving a boost with DNA, sE1E2 protein or the VLPs ( Figure 4B).…”

“…To assess the neutralizing ability of the antibodies generated by the different vaccination regimens, we investigated the ability of the sera to inhibit binding of genotype 1b HCV VLPs to Huh7 cells. Fluorescent-labeled HCV VLPs were incubated with the serum at 1/5 and 1/25 dilutions, and the binding of the VLPs to Huh7 cells was monitored by flow cytometry as described (66,67). Incubation of the HCV VLPs with PBS (VLP only) or normal mouse serum (NMS; Sigma) resulted in minimal inhibition of HCV VLP binding to hepatocytes (8%), whereas the addition of anti-CD81 antibodies on the other hand (Figure 6), inhibited VLP binding to Huh7 cells by >90% confirming that these particles use CD81 to bind to target cells, in a similar manner to HCV (74,75).…”

Progress in Vaccine Development for HCV Infection

“…Challenging with infectious HCV, one chimpanzee developed transitory viremia, and the other three displayed higher levels of viremia, but after 10 weeks, their viral levels became immeasurable as reported by Elmowalid et al [41]. Technique for high-capacity purification of HCV VLPs was defined by Earnest-Silveira et al [42]. The structural HCV protein coding sequences of genotypes 1a, 1b, 2a, or 3a were coexpressed using a recombinant adenoviral expression system in Huh7 cell line.…”